UNIVERSITI PUTRA MALAYSIA
WAEL MOUSA
FSTM 2012 15
AN INTEGRATED MODELLING APPROACH TO POSTHARVEST CONTROL OF Aspergillus flavus GROWTH AND AFLATOXIN
PRODUCTION IN PADDY GRAINS AND RICE
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AN INTEGRATED MODELLING APPROACH TO POSTHARVEST
CONTROL OF Aspergillus flavus GROWTH AND AFLATOXIN
PRODUCTION IN PADDY GRAINS AND RICE
By
WAEL MOUSA
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfilment of the Requirements for the Degree of Doctor of Philosophy
May 2012
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
of the requirements for the degree of Doctor of Philosophy
AN INTEGRATED MODELLING APPROACH TO POSTHARVEST
CONTROL OF Aspergillus flavus GROWTH AND AFLATOXIN
PRODUCTION IN PADDY GRAINS AND RICE
ABSTRACT
By
WAEL MOUSA
May 2012
Chairman: Farinazleen Mohammed Ghazali, PhD
Faculty: Food Science and Technology
The main aim of the present study was to develop an integrated postharvest strategy
for controlling the growth of and aflatoxin production by Aspergillus flavus on
paddy and rice. Initially, equilibrium moisture content of paddy was studied at 20,
30, 40 and 50°C at relative humidity (RH) between 0.113 and 0.976 using
gravimetric technique. The isotherms displayed the general sigmoidal curve Type II
and exhibited hysteresis where it was more pronounced at lower temperatures. At
fixed RH, the sorption characteristics were temperature-dependent where the
sorption capacity of the paddy increased as the temperature was decreased. Among
the models assessed for their ability to fit the sorption data, the Oswin equation was
the best followed by the third order polynomial, GAB, Smith, Chung-Pfost, and
Henderson models. Therefore, the Oswin model was chosen to estimate the amount
of water required to rehydrate dried paddy to the desired water activity (aw) in
subsequent studies. Thereafter, the growth of two aflatoxigenic A. flavus on paddy
and aflatoxin production were studied following a full factorial design with seven aw
levels between 0.82 and 0.99 and seven temperatures between 10 and 43°C. The
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growth of the fungi, expressed as colony diameter (mm), was measured daily and
aflatoxin production was analyzed using isocratic HPLC with a fluorescence
detector. The maximum colony growth rates of both isolates were estimated by
fitting the primary model of Baranyi to growth data. Three potentially suitable
secondary models; Rosso, polynomial, and Davey, were assessed for their ability to
describe the radial growth rate as a function of temperature and aw. Both strains
failed to grow at the marginal temperatures (10 and 43°C) regardless of the aw
studied, and at the aw level of 0.82, regardless of the temperature. Despite that the
predictions of all studied models showed good agreement with the observed growth
rates, the Davey model proved to be the best predictor of the experimental data.
Aflatoxins were detected at aw between 0.86-0.99 with an optimal aw of 0.98 and the
optimal temperature was in the range of 25-30°C. Then, the effect of aw (0.82-0.92)
and temperature (15-42°C) on the growth and aflatoxin production by A. flavus on
polished and brown rice was also studied. Four secondary models were used to
implicit the combined effects of aw and temperature on the growth rates. All models
were validated using independent experimental data. According to the assessment
indices, the performance of the Davey model in describing the experimental data was
the highest, followed in decreasing order by the polynomial, Gaussian and Rosso
models. The estimated optimal growth temperature was between 30-34°C. Neither
growth nor aflatoxins were detected at aw 0.82 on polished rice while growth and
aflatoxins were detected at this aw between 25-35°C on brown rice. The highest
amounts of aflatoxins were formed at the higher aw values (0.90-0.92) and a
temperature of 20°C after 21 days of incubation for both types of rice where the
consistency of aflatoxin production within a wider range of aw values occurred
between 25-30°C. The results also showed that brown rice tended to support higher
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A. flavus growth and aflatoxin production than the polished rice. Logistic models
describing the growth and aflatoxin production boundaries of A. flavus were also
developed. Experiments were conducted at aw between 0.80 and 0.99 and
temperature between 10 °C and 45°C on rice meal agar (RA) for duration of four
weeks. The degree of agreement between the predicted and observed data in terms of
concordance was > 97% and > 98% for growth and aflatoxin production,
respectively. Probabilities of growth and aflatoxin production at 21 days were almost
equal to those at 28 days. The polynomial logistic models that were developed were
validated with data obtained from repeated experiments on paddy. The models were
successfully able to predict the probabilities with concordance rates of 85.2% and
88.9% for growth and aflatoxin production, respectively, whereas all the
misidentified cases were found to be false positive. Then, the potential of modified
atmosphere packaging with 20-80% CO2 (balanced with nitrogen) in controlling the
growth and aflatoxin production on paddy at different aw (0.92-0.98) relative to the
control (0% CO2) was examined using the two above-mentioned fungal isolates.
Except at 0.92 aw, as much as 80% CO2 failed to inhibit the growth of the fungi
completely. However, at all aw levels studied, the growth parameters as estimated by
Baranyi function and aflatoxin production were affected by the increment in CO2
where growth rate and aflatoxin production were negatively correlated with CO2
while the lag phase correlated positively with CO2. At 0.98 aw, atmosphere enriched
with 20% and 80% CO2 led to at least 59% and 88% reduction in growth and 47%
and 97% in aflatoxin production, respectively. At 0.95 aw, the lag phases of both
isolates on average increased by a factor of 1.7-12.0 when the CO2 levels in the
headspace were between 20-80% compared to the control. Finally, the effectiveness
of three essential oils (cinnamon, glove and thyme) and three antioxidants [butylated
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hydroxyanisole (BHA), propyl paraben (PP) and butylated hydroxytoluene (BHT)]
on controlling the growth of and aflatoxin formation by the A. flavus grown on rice
meal agar and paddy grains at different aw (0.92, 0.95, and 0.98) were evaluated.
Two of the antioxidants (BHA and PP) and the three essential oils displayed
significant inhibitory effect on the growth and aflatoxin formation on in vitro and on
the paddy grains. Regardless of aw of the paddy, the application of essential oils at
500 µg g-1
reduced the growth and aflatoxin formation by > 55% and >80%,
compared with >75% and > 82% with usage of antioxidants (BHA, PP),
respectively. The estimated effective doses 50% (ED50) required to reduce growth
and aflatoxin formation on rice meal agar were lower than their counterpart on paddy
and those required to inhibit aflatoxin formation were lower those of growth.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Doktor Falsafah
PENDEKATAN MODEL BERSEPADU KAWALAN LEPAS TUAI PADI
DAN BERAS DARI PERTUMBUHAN Aspergillus flavus DAN
PENGHASILAN AFLATOKSIN
Oleh
WAEL MOUSA
Mei 2012
Pengerusi: Farinazleen Mohammed Ghazali, PhD
Fakulti: Fakulti Sains Dan Teknologi Makanan
ABSTRAK
Tujuan utama kajian semasa adalah untuk membina strategi lepas tuai bersepadu
untuk mengawal pertumbuhan dan penghasilan aflatoksin oleh Aspergillus flavus
pada padi dan beras. Permulaannya, kandungan kelembapan keseimbangan padi
dikaji pada 20, 30, 40 dan 50 °C pada kelembapan relatif (RH) di antara 0.113 dan
0.976 menggunakan teknik gravimetrik. Isoterma menunjukkan lengkungan
sigmoidal Jenis II am dan mempamerkan histerisis di mana ia adalah lebih ketara
pada suhu lebih rendah. Pada RH tetap, ciri penyerapan adalah suhu-kebergantungan
di mana kapasiti penyerapan padi meningkat apabila suhu menurun. Antara model-
model yang dinilai untuk keupayaan mereka yang sesuai dengan data penyerapan,
persamaan Oswin adalah yang terbaik diikuti dengan model-model polinomial tertib
ketiga, GAB, Smith, Chung-Pfost dan Henderson. Oleh itu, model Oswin telah
dipilih untuk menganggar kandungan air diperlukan untuk menghidrat semula padi
kering kepada aktiviti air (aw) diingini dalam kajian selanjutnya. Berikutnya,
pertumbuhan dua aflatoksigenik A. flavus pada padi dan penghasilan aflatoksin
dikaji mengikut reka bentuk faktorial penuh dengan tujuh tahap aw di antara 0.82 dan
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0.99 dan tujuh suhu di antara 10 dan 43 °C. Pertumbuhan kulat, dinyatakan sebagai
diameter koloni (mm), diukur setiap hari dan penghasilan aflatoksin telah dianalisis
menggunakan HPLC isokratik dengan pengesan pendafluoran. Kadar pertumbuhan
koloni yang maksimum daripada kedua-dua isolat telah dianggarkan dengan
menyesuaikan model utama Baranyi kepada data pertumbuhan. Tiga model sekunder
berpotensi yang sesuai; Rosso, polinomial dan Davey, telah dinilai keupayaan
mereka untuk menerangkan kadar pertumbuhan radial sebagai fungsi suhu dan aw.
Kedua-dua strain gagal untuk tumbuh pada suhu marginal (10 dan 43 °C) tanpa
mengira aw yang dikaji, dan pada tahap aw 0.82, tanpa mengira suhu. Meskipun
ramalan kesemua model yang dikaji menunjukkan persetujuan yang baik dengan
kadar pertumbuhan yang diperhatikan, model Davey terbukti sebagai peramal terbaik
untuk data eksperimentasi. Aflatoksin telah dikesan pada aw di antara 0.86-0.99
dengan aw optimal pada 0.98 dan suhu optimal dalam lingkungan 25-30 °C.
Kemudian, kesan aw (0.82-0.92) dan suhu (15-42 °C) pertumbuhan dan penghasilan
aflatoksin oleh A. flavus pada beras putih dan beras perang juga dikaji. Empat model
sekunder telah digunakan secara mutlak untuk kesan gabungan aw dan suhu terhadap
kadar pertumbuhan. Kesemua model telah disahihkan menggunakan data
eksperimentasi bebas. Berpandukan kepada indeks penilaian, perlaksanaan mmodel
Davey dalam menghuraikan data eksperimentasi adalah yang tertinggi, diikuti dalam
susunan menurun oleh model-model polinomial, Gaussian dan Rosso. Anggaran
suhu pertumbuhan optima adalah di antara 30-34 °C. Tiada pertumbuhan mahupun
aflatoksin telah dikesan pada aw 0.82 pada beras putih manakala pertumbuhan dan
aflatoksin telah dikesan pada aw di antara 25-35 °C pada beras perang. Jumlah
aflatoksin yang tertinggi telah dibentuk pada nilai aw (0.90-0.92) yang lebih tinggi
dan suhu 20 °C selepas 21 hari inkubasi untuk kedua-dua jenis beras di mana
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penghasilan aflatoksin secara konsisten di dalam julat nilai aw yang lebih luas
berlaku di antara 25-30 °C. Keputusan juga menunjukkan bahawa beras perang
cenderung untuk menyokong pertumbuhan A. flavus dan penghasilan aflatoksin yang
lebih tinggi berbanding beras putih. Model logistik yang menghuraikan sempadan
pertumbuhan dan penghasilan aflatoksin A. flavus juga telah dibangunkan.
Eksperimen telah dijalankan pada aw di antara 0.80 dan 0.99 dan suhu di antara 10
°C dan 45 °C pada agar tepung beras (RA) untuk jangka masa empat minggu. Darjah
persetujuan di antara data ramalan dan pemerhatian dalam terma kesejajaran adalah
> 97% dan > 98% untuk untuk pertumbuhan dan penghasilan aflatoksin, masing-
masing. Kebarangkalian pertumbuhan dan penghasilan aflatoksin pada 21 hari
adalah hampir sama dengan kebarangkalian pada 28 hari. Model-model logistik
polinomial yang telah dibangunkan telah disahihkan dengan data yang diperolehi
daripada eksperimen ulangan terhadap padi. Model-model tersebut dengan
berjayanya mampu meramal kebarangkalian dengan kadar kesejajaran 85.2% dan
88.9% untuk pertumbuhan dan penghasilan aflatoksin, masing-masing. Manakala
kesemua kes salah pengesanan telah didapati sebagai positif palsu. Kemudian,
potensi pembungkusan atmosfera terubah suai dengan 20-80% CO2 (diseimbangkan
dengan nitrogen) dalam mengawal pertumbuhan dan penghasilan aflatoksin terhadap
padi pada aw (0.92-0.98) yang berbeza relatif kepada kawalan (0% CO2) telah
diselidik menggunakan dua isolat kulat yang dinyatakan di atas. Kecuali pada aw
0.92, CO2 sebanyak 80% gagal untuk merencat pertumbuhan kulat sepenuhnya.
Bagaimanapun, pada kesemua tahap aw yang dikaji, parameter-parameter
pertumbuhan seperti yang dianggar dengan fungsi Baranyi dan penghasilan
aflatoksin telah dipengaruhi dengan peningkatan dalam CO2 di mana kadar
pertumbuhan dan penghasilan aflatoksin adalah berhubung kait secara negatif
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dengan CO2 manakala fasa lag berhubung kait secara positif dengan CO2. Pada aw
0.98, atmosfera yang diperkaya dengan 20% dan 80% CO2 membawa kepada
sekurang-kurangnya 59% dan 88% pengurangan dalam pertumbuhan dan 47% dan
97% dalam penghasilan aflatoksin, masing-masing. Pada aw 0.95, fasa-fasa lag
kedua-dua isolat secara purata meningkat dengan faktor 1.7-12.0 apabila tahap CO2
di dalam ruang kepala adalah di antara 20-80% berbanding kawalan. Akhirnya,
keberkesanan tiga minyak esen (kayu manis, bunga cengkih dan thyme) dan tiga
antioksidan [butylated hidroksianisol (BHA), propyl paraben (PP) dan butylated
hidroksitoluena (BHT)] dalam mengawal pertumbuhan dan pembentukan aflatoksin
oleh A. flavus yang dibiakkan pada agar tepung beras dan bijirin padi pada aw (0.92,
0.95 dan 0.98) yang berbeza telah dinilai. Dua daripada antioksidan (BHA dan PP)
dan ketiga-tiga minyak esen mempamerkan kesan perencatan yang signifikan
terhadap pertumbuhan dan pembentukan aflatoksin pada in vitro dan pada bijirin
padi. Tanpa mengira aw pada padi, penggunaan minyak-minyak esen pada 500 μg g-1
mengurangkan pertumbuhan dan pembentukan aflatoksin dengan > 55% dan > 80%,
berbanding dengan > 75% dan > 82% dengan pengunaan antioksidan (BHA, PP),
masing-masing. Anggaran 50% dos-dos berkesan (ED50) yang diperlukan untuk
mengurangkan pertumbuhan dan pembentukan aflatoksin pada agar tepung beras
adalah lebih rendah berbanding yang digunakan pada padi dan yang diperlukan
untuk merencatkan pertumbuhan aflatoksin adalah lebih rendah berbanding yang
diperlukan untuk pertumbuhan.
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ACKNOWLEDGEMENTS
“In the Name of Allah, the Most Merciful and the Most Beneficent”
All praise do to allah, Lord of the universe. Only by his grace and mercy this thesis
was completed. First and for most I would like to express my profound gratitude to
my honourable supervisor Dr. Farinazleen Mohamad Ghazali for her invaluable
advice, guidance, encouragement. I would like to wish her unending success in her
family and professional lives. I am extremely grateful to my supervisory committee
members, Prof. Dr. Jinap Selamat, Pof. Dr. Hasanah Mohd. Ghazali and Prof. Dr.
Son Radu for their valuable contribution and suggestions.
I am grateful to my colleagues in the laboratory of food safety and quality; Kabir
Umar, Elham Farahany, Sahar Arzandeh, Gisia Daniali, Wendy Lim, Diyana
Syamim, Afsaneh Farhadian, Dr. Parvaneh Hajeb for being supportive friends and
wish them all the very best for the future. I am indebted to the staff of the Faculty of
Food Science and Technology, University Putra Malaysia who assisted me in one
way or the other. Special thanks to Norliza Othman for her assistance through my
study.
I would like to express my sincere gratitude to my parents, brothers and sisters for
their prayers, unending encouragement and continuous moral support. Last but not
least, my beloved wife, Myssa Mousa and my dear kids (Abdulrahman and
Abdullah) deserve my deepest and heartfelt gratitude for their bearing with me for
the many months I spent away from home.
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I certify that a Thesis Examination Committee has met on 15 May 2012 to conduct
the final examination of Wael A. S. Mousa on his thesis entitled “An Integrated
Modelling Approach to Postharvest Control of Aspergillus flavus Growth and
Aflatoxin Production in Paddy Grains and Rice” in accordance with the Universities
and University Colleges Act 1971 and the Constitution of the Universiti Putra
Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the
student be awarded the Doctor of Philosophy.
APPROVAL
Members of the Thesis Examination Committee were as follows:
Mohd Yazid bin Abd Manap, PhD
Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Chairman)
Fatimah binti Abu Bakar, PhD
Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Internal Examiner)
Cheah Yoke Kqueen, PhD
Associate Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Internal Examiner)
R. Russell M. Paterson, PhD
Professor
Centre of Biological Engineering
University of Minho
Portugal
(External Examiner)
SEOW HENG FONG, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 28 June 2012
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfillment of the requirement for the degree of Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Farinazleen Mohamad Ghazali, PhD
Associate Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Chairman)
Jinap Selamat, PhD
Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Member)
Hasanah Mohd. Ghazali, PhD
Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Member)
Son Radu, PhD
Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Member)
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 28 June 2012
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which
have been duly acknowledged. I also declare that it has not been previously, and is
not concurrently, submitted for any other degree at Universiti Putra Malaysia or at
any other institution.
WAEL MOUSA
Date: 15 May 2012
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TABLE OF CONTENTS
Page
ABSTRACT i
ABSTRAK v
ACKNOWLEDGEMENTS ix
APPROVAL x
DECLARATION xii
LIST OF TABLES xviii
LIST OF FIGURES xxi
LIST OF ABBREVIATIONS
xxv
CHAPTER
1
INTRODUCTION
1
1.1 Specific objectives 4
2
LITERATURE REVIEW 6
2.1 Introduction 6
2.2 Water availability 7
2.3 Moisture sorption isotherm 9
2.4 Factor affecting fungal growth in the grains
ecosystem
9
2.4.1 Effect of water activity on fungal growth 10
2.4.2 Effect of temperature on fungal growth 11
2.4.3 Fungal interaction in grain ecosystem 12
2.4.4 Effect of inter-gaseous composition on fungal
growth
13
2.4.5 Relationship between insects manifestation
and fungi in grain ecosystem
14
2.5 Possible effect of climate change on change on the
growth of mycotoxingenic fungi
15
2.6 Mycotoxins 16
2.6.1 Aflatoxins 17
2.6.2 Aflatoxin analysis 24
2.7 Control of fungal growth and aflatoxins 29
2.7.1 Control of aflatoxigenic fungi using modified
atmosphere storage
29
2.7.2 Control of aflatoxigenic fungi using
antioxidants
30
2.7.3 Control of aflatoxigenic fungi using essential
oils
31
2.7.4 Biological control of aflatoxigenic fungi 34
2.8 Effect of milling on aflatoxins level in rice 36
2.9 Predictive mycology 38
2.9.1 Primary models 39
2.9.2 Secondary models 42
2.9.3 Tertiary models 46
2.9.4 Probability models 46
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3 SORPTION ISOTHERMS AND ISOSTERIC HEATS
OF SORPTION OF MALAYSIAN PADDY
48
3.1 Introduction 48
3.2 Materials and methods 50
3.2.1 Materials 50
3.2.2 Experimental procedure 50
3.2.3 Fitting sorption data to various isotherm
equations
51
3.2.4 Determination of the net isosteric heat of
sorption
52
3.3 Results 53
3.3.1 Moisture sorption isotherm and sorption
hysteresis
53
3.3.2 Modelling the sorption isotherm 56
3.3.3 Isosteric heat of sorption 62
3.4 Discussion 63
3.4.1 Moisture sorption isotherm and sorption
hysteresis
63
3.4.2 Modelling sorption isotherm 65
3.4.3 Isosteric heats of sorption 67
3.5 Conclusion 68
4
MODELLING THE EFFECT OF WATER ACTIVITY
AND TEMPERATURE ON THE GROWTH RATE OF
AND AFLATOXIN PRODUCTION BY Aspergillus flavus
IN PADDY
69
4.1 Introduction 69
4.2 Materials and methods 72
4.2.1 Fungal isolates 72
4.2.2 Experimental design 72
4.2.3 Preparation and inoculation of paddy grains 73
4.2.4 Assessment of fungal growth 74
4.2.5 Modelling fungal growth as a function of aw
and temperature
74
4.2.6 Modelling aflatoxin production as a function
of aw and temperature
76
4.2.7 Model validation 77
4.2.8 Determination of aflatoxin 77
4.3 Results 79
4.3.1 Effect of aw and temperature on the growth
rate of A. flavus on paddy
79
4.3.2 Model validation 87
4.3.3 Effect of aw and temperature on Aflatoxin
production by A. flavus on paddy
91
4.4 Discussion 94
4.5 Conclusion 98
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5 EFFECT OF WATER ACTIVITY AND
TEMPERATURE ON THE GROWTH AND
AFLATOXIN PRODUCTION BY Aspergillus flavus ON
POLISHED AND BROWN RICE: DEVELOPMENT
AND ASSESSMENT OF DIFFERENT GROWTH
MODELS
100
5.1 Introduction 100
5.2 Materials and methods 101
5.2.1 Fungal isolates 101
5.2.2 Experimental design 101
5.2.3 Preparation of rice 102
5.2.4 Inoculation, incubation and growth
assessment
102
5.2.5 Model development 103
5.3 Results 107
5.3.1 Modelling the growth rate as a function of
temperature and aw
107
5.3.2 Validation and evaluation of performance of
the models
113
5.3.3 Effect of temperature, aw, and incubation time
on the formation of aflatoxins
5.4 Discussion 116
5.5 Conclusion 119
6
MODELLING GROWTH/NO GROWTH AND
AFLATOXIN PRODUCTION BOUNDARY OF
Aspergillus flavus ON RICE
125
6.1 Introduction 125
6.2 Materials and methods 126
6.2.1 Experimental design 126
6.2.2 Fungal isolates 127
6.2.3 Preparation of the medium 127
6.2.4 Inoculum preparation, inoculation,
incubation, and growth monitoring
127
6.2.5 Determination of aflatoxins 128
6.2.6 Modelling growth no /growth and aflatoxins
production boundaries
129
6.2.7 Model validation 130
6.3 Results 131
6.3.1 Modelling growth/no growth interface 131
6.3.2 Modelling aflatoxins production/no
production interface
138
6.3.3 Model validation 144
6.4 Discussion 145
6.5 Conclusion 151
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7 TEMPERATURE, WATER ACTIVITY AND GAS
COMPOSITION EFFECTS ON THE GROWTH AND
AFLATOXIN PRODUCTION BY Aspergillus flavus ON
PADDY
152
7.1 Introduction 152
7.2 Materials and methods 153
7.2.1 Experimental design 153
7.2.2 Fungal isolates 153
7.2.3 Influence of temperature and water activity
on the relation between colony diameters and
aflatoxins production on paddy
154
7.2.4 Influence of modified atmosphere packaging
(MAP) on the growth and aflatoxin
production on paddy with different water
activity
154
7.2.5 Determination of aflatoxins 155
7.2.6 Mathematical and statistical analysis 155
7.3 Results 157
7.3.1 Effect of temperature and aw on the relation
between colony diameter and aflatoxins
production by A. flavus on paddy
157
7.3.2 Effects of aw and initial headspace CO2 on the
growth parameters of A. flavus on paddy
160
7.3.3 Effects of aw and initial headspace CO2 on
aflatoxins production in paddy
164
7.4 Discussion 165
7.5 Conclusion 170
8 EFFICACY OF ESSENTIAL OILS AND
ANTIOXIDANTS IN CONTROLLING OF GROWTH
AND AFLATOXIN PRODUCTION BY Aspergillus flavus
ON PADDY
171
8.1 Introduction 171
8.2 Materials and methods 172
8.1.2 Screening study for the effect of essential oil
growth of A. flavus
172
8.2.2 In vitro effect of essential oils and antioxidant
on the growth and aflatoxin production by A.
flavus
173
8.2.3 Effect of essential oils and antioxidant on the
growth and aflatoxin production by A. flavus
on paddy
174
8.2.3 Aflatoxin analysis 175
8.2.3 Mathematical and statistical analysis 175
8.3 Results 176
8.3.1 Screening study for the effect of essential oil
growth of A. flavus
176
8.3.2 In vitro effect of essential oil and antioxidants
on the growth of and aflatoxin production by
A. flavus
176
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8.3.3 Effect of essential oil on the growth of and
aflatoxin production by A. flavus on paddy
grains
184
8.4 Discussion 191
8.5 Conclusion 194
9 SUMMARY, CONCLUSION AND
RECOMMENDATION FOR FUTURE RESEARCH
195
9.1 Summary and conclusion 195
9.2 Recommendation for future research
202
REFERENCES 203
BIODATA OF STUDENT 229
LIST OF PUBLICATIONS 230